Genetically modified cyclic-nucleotide controlled ion channels and the use thereof

ABSTRACT

The present invention relates to genetically modified cyclic nucleotide-gated ion channels whose subunits have been altered so as to have higher sensitivity to cAMP and/or higher selectivity for cAMP compared to cGMP in comparison with the wild type according to SEQ ID NO 1 and 2.

This application is a 371 of PCT/EP02/08756 filed Aug. 06, 2002.

The present invention relates to genetically modified nucleic acids,preferably DNAs, which code for cyclic nucleotide-gated ion channels(CNG channels) and the corresponding proteins and the use thereof.

Chemical substances such as, for example, hormones or neurotransmittersmay bind as “primary messengers” (ligands) to the membrane of cells andthereby trigger a large variety of biochemical-physiological reactionsinside said cells, which enable the latter to respond to theirenvironment. This process is mediated by a large number ofmembrane-bound receptors to which ligands can bind specifically anddirectly. It is at the beginning of different, partly extremely complextransaction cascades. Receptors control and regulate via such cascadesthe activity of different cellular proteins (effector proteins). Theseeffector proteins for their part can in turn regulate the concentrationof intracellular messengers, the “secondary messengers”. Only suchsecondary messengers control a multiplicity of physiological reactionssuch as, for example, synthesis and release of hormones andneurotransmitters, cell division and cell growth and excitation andexcitability of neuronal cells. Particularly important secondarymessengers include cyclic adenosine monophosphate (cAMP), cyclicguanosine monophosphate (cGMP) and Ca²⁺ ions.

The intracellular concentration of secondary messengers is usuallyregulated by signal transduction cascades in which G protein-coupledreceptors in the membrane of cells (GPCRs) register an extracellularsignal then activate corresponding G proteins which in turn eitherstimulate or inhibit the activity of the corresponding effector proteins(Morris A. J. and Malbon C. C. (1999) Physiological regulation of Gprotein-linked signaling. Physiol. Rev., 79, 1373-1430). In addition,other proteins can modulate the activity of each individual componentwithin the framework of a large variety of feedback mechanisms.

Deviations from the physiologically normal concentration of ligands ordisruptions in the course of a signal transduction cascade, caused, forexample, by the malfunction of a component involved therein, may causesevere diseases. Since GPCRs represent a particularly importantinterface between the extracellular and intracellular medium of the cellby serving as binding site or site of attack of a very large number ofendogenous and exogenous chemical substances and, moreover, controllingin a large variety important physiological processes in virtually anyvital organ or tissue, they are of outstanding interest formedical-pharmacological interventions. Particularly important areas ofindication in this connection are disorders of the central andperipheral nervous system, of the cardiovascular system and the innerorgans.

A therapeutic goal of the pharmaceutical industry is to developpharmacological active compounds which activate (agonists), inhibit(antagonists) the target proteins or else modulate the activity thereof.This additionally requires detailed functional characterization of theappropriate target proteins. For this purpose, different methods havebeen developed in recent years, which differ, some of them markedly,with respect to their flexibility, but also to the meaningfulness of theresults obtained therewith, and to their robustness and theireffectiveness regarding speed, amount of work required and costs.

Now that the complementary DNA of a multiplicity of receptors have beencloned and said receptors can be expressed functionally in cell systems,the studies are carried out mainly on heterologously expressedreceptors. The prior art regarding the strategies and methods used andapplication thereof are described, for example, in articles by HertzbergR. P. and Pope A. J. (2000) High-throughput screening: new technologyfor the 21st century. Curr. Opin. Chem. Biol., 4, 445-451, Howard A. D.,MacAllister G., Feighner S. D., Liu Q., Nargund R. P., van der Ploeg L.H., and Patchett A. A. (2001) Orphan G-protein-coupled receptors andnatural ligand discovery. Trends Pharmacol. Sci., 22, 132-140, CivelliO., Northacker H. P., Saito Y., Wang Z., Lin S. H. and Reinscheid R. K.,(2001) Novel neuro-transmitters as natural ligands of orphanG-protein-coupled receptors. Trends Neurosci., 24, 230-237, and also inthe references contained therein.

More than 60% of the GPCRs known regulate the intracellularconcentration of the secondary messenger cAMP. Different methods existfor studying the effect of chemical substances on such GPCRs and thecorresponding effector proteins.

Some of these methods are based on direct, usually radiochemical,measurements of intracellular cAMP concentration. For this purpose, forexample, the cells are stimulated, biochemically disrupted after adefined period of time and the change in cAMP concentration isdetermined. Although these measurement methods are very sensitive, theyare usually inherently slow, cost-intensive and time-consuming. It is,moreover, not possible to monitor the change in intracellular cAMPconcentration in real time. Important characteristic properties such asspeed and course of an activation or inhibition can be determined onlyby a multiplicity of additional measurements at considerable additionalexpense. On the other hand, said methods are advantageous in that it ispossible to study the effect of active compounds not only on GPCRs butalso on the effector proteins which regulate intracellular cAMPconcentration.

Another method which may be used for measuring intracellular changes incAMP or cGMP concentration makes use of the properties of membrane-boundCNG channels. Cyclic nucleotide-gated ion channels (CNG channels) aremembrane-bound proteins which have the features and properties describedbelow (Finn J. T., Grunwald M. E. and Yau K. W. (1996) Cyclicnucleotide-gated ion channels: an extended family with diversefunctions. Annu. Rev. Physio., 58, 395-426; Richards M. J. and Gordon S.E. (2000) Cooperativity and cooperation in cyclic nucleotide-gated ionchannels. Biochemistry, 39, 14003-14011). CNG channels comprise (1)presumably 4 or 5 subunits (α and/or β subunits) which (2) in each casespan the membrane six times and (3) possess in each case a binding sitefor cyclic nucleotides at the carboxy-terminal intracellular end. CNGchannels are (4) activated directly and in a manner dose-dependent bycAMP or cGMP, form (5) an aqueous pore in the membrane with aconductivity which is only slightly selective for monovalent cations andare (6) likewise permeable for divalent cations such as Ca²⁺ ions, forexample.

“Binding site for cyclic nucleotides” refers to that section in the CNGchannel subunits to which the cyclic nucleotides cAMP and cGMP can bindin a dose-dependent manner. The amino acid sequence in this sectiondetermines to a considerable extent the sensitivity of a CNG channel forcAMP or cGMP (sensitivity).

“Conductivity” refers to the property of ion channels of enabling in amore or less selective manner ions to flow from the outside of the cellinto the cell interior or to flow out of the cell interior to theoutside. For this purpose, the ion channels form an opening in themembrane (aqueous pore), through which, depending on the state ofactivation of said channels, ions can flow in or out, according to theconcentration gradient.

It is possible in heterologous expression systems to express functionalCNG channels from identical α subunits (homooligomers; Kaupp U. B.,Niidome T., Tanabe T., Terada S., Bönigk W., Stühmer W., Cook N. J.,Kanagawa K., Matsuo H., Hirose T., Miyata T. and Numa S. (1989) Primarystructure and functional expression from complementary DNA of the rodphotoreceptor cyclic GMP-gated channel. Nature, 342, 762-766), fromdifferent a subunits (heterooligomers; Bradley J., Li J., Davidson N.,Lester H. A. and Zinn K. (1994) Heteromeric olfactory cyclicnucleotide-gated channels: a subunit that confers increased sensitivityto cAMP, Proc. Natl. Acad. Sci. USA, 91, 8890-8894; Liman E. R. and BuckL. B. (1994) A second subunit of the olfactory cyclic nucleotide-gatedchannel confers high sensitivity to cAMP. Neuron 13, 611-621), and asheterooligomers from α and β subunits (Chan T. Y., Peng Y. W., DhallanR. S., Ahamed B., Reed R. R. and Yau K. W. (1993) A new subunit of thecyclic nucleotide-gated cation channel in retinal rods. Nature, 362,764-767). The β subunits alone cannot form functional channels but haveexclusively modulatory functions in heterooligomeric CNG channels (ChenT. Y., Peng Y. W., Dhallan R. S., Ahamed B., Reed R. R. and Yau K. W.(1993) A new subunit of the cyclic nucleotide-gated cation channel inretinal rods. Nature, 362, 764-767). When cAMP or cGMP binds to CNGchannels, said channels open in a dose-dependent manner and ions flowinto the cell. Activation of the CNG channels results underphysiological conditions in an increased Ca²⁺ conductivity of saidchannels and thus causes the increase in intracellular Ca²⁺concentration. A change in concentration of this kind can be measuredusing optical Ca²⁺ measurement methods. Thus, these ion channels couldin principle be used as cellular cAMP sensor for studying andcharacterizing any receptors and intracellular proteins which regulateintracellular cAMP concentration. This method is very rapid, effectiveand inexpensive in comparison with direct cAMP measurements. It allows ahigh throughput of tests per day and makes real-time measurementspossible. This method is therefore in principle particularly suitablefor pharmacological drug screening.

The documents U.S. Pat. No. 6,001,581 and WO 98/58074 and Gotzes F.(1995) Dissertation. ISSN 0944-2952 describe the use as cAMP sensor ofCNG channels comprising the α3 subunits from the epithelium of the nose.However, such CNG channels have several decisive disadvantages when usedas cellular cAMP sensors in pharmaceutical drug screening. As little as2 μM cGMP activates these CNG channels but only 80 μM cAMP produceshalf-maximum activation thereof (Dhallan R. S., Yau K. W., Schrader K.A. and Reed R. R. (1990) Primary structure and functional expression ofa cyclic nucleotide-activated channel from olfactory neurons. Nature,347, 184-187; Ludwig J., Margalit T., Eismann E., Lancet D. and Kaupp U.B. (1990) Primary structure of cAMP-gated channel from bovine olfactoryepithelium. FEBS Lett. 270, 24-29). However, since intracellular cAMPconcentration usually changes only by a few μM, such CNG channels areonly poorly suitable as cAMP sensors, although they are suitable as cGMPsensors in principle. Moreover, even small fluctuations in intracellularcGMP concentration can interfere with the cAMP concentrationmeasurements.

In contrast, half-maximum activation (K_(1/2) value) of heterooligomericCNG channels composed of α3, α4 and β1b subunits is already obtained ata cAMP concentration of about 4 μM, while K_(1/2) for cGMP changes onlyinsignificantly in comparison with the homooligomeric channels (BönigkW., Bradley J., Müller F., Sesti F., Boekhoff I., Ronnett G. V., KauppU. B and Frings S., (1999) The native rat olfactory cyclicnucleotide-gated channel is composed of three distinct subunits. J.Neurosci., 19, 5332-5347. CNG channels of this kind have in principleexcellent suitability as cellular cAMP sensors. Disadvantageously,however, expression of such channels in heterologous cell systemsrequires a lot of work and time. Moreover, small fluctuations inintracellular cGMP concentration may interfere with the cAMP sensorfunction.

However, it is also possible to use molecular-biological methods forpreparing CNG channels which comprise only α subunits but arenevertheless highly sensitive to cAMP: in 1991, a genetically modifiedα3 subunit of the bovine CNG channel was described, in which subunitthreonine at position 537 had been replaced with a serine (T537S)(Altenhofen W., Ludwig J., Eismann E., Kraus W., Bönigk W. and Kaupp U.B. (1991) Control of ligand specificity in cyclic nucleotide-gatedchannel from rod photoreceptors and olfactory epithelium. Proc. Natl.Acad. Sci. USA, 88, 9868-9872). This subunit forms CNG channels whosehalf-maximum activation is produced by 14 μM cAMP. Threonine T537 islocated in the sequence section of the α3 subunit, which is involved toa considerable extent in binding of the cyclic nucleotides. Evidently,the amino acid in this position is particularly important for thesensitivity of the CNG channels (Altenhofen W., Ludwig J., Eismann E.,Kraus W., Bönigk W. and Kaupp U. B. (1991) Control of ligand specificityin cyclic nucleotide-gated channel from rod photoreceptors and olfactoryepithelium. Proc. Natl. Acad. Sci. USA, 88, 9868-9872). However, saidmutation, T537S, also increases the sensitivity of the channels to cGMP(K_(1/2)=0.7 μM). Such channels (T537S mutants) can be expressedheterologously with low expenditure, but they are, as cAMP sensor, evenmore susceptible to interference from small fluctuations inintracellular cGMP concentration than the heterooligomeric channels.Moreover, the sensitivity to cAMP is still not high enough in order toreliably register also small fluctuations in intracellular cAMPconcentration.

Furthermore, mutations in the α3 subunit of the CNG channel are knownwhich increase sensitivity to cAMP and additionally reduce sensitivityto cGMP. (Rich T. C., Tse T. E., Rohan D. G., Schaack J. and Karpen J.W. (2001) In vivo assessment of local phosphodiesterase activity usingtailored cyclic nucleotide-gated channels as cAMP sensors. J. Gen.Physiol., 118; 63-78). 1.2 μM cAMP but only 12 μM cGMP producehalf-maximum activation of CNG channels composed of the rat α3 subunitin which cysteine in position 460 (C460) has been replaced withtryptophan (W) and, in addition, glutamate in position 583 (E583) hasbeen replaced with methionine (M) (C460W/E583M mutant).

It was the object of the invention to develop CNG channels as cAMPsensors whose sensitivity to cAMP or cGMP is similar to that of theC460W/E583M mutant but which are genetically modified only in oneposition. Such CNG channels may be used in simple and rapid cellularmeasuring systems efficiently and universally for pharmaceutical drugscreening but also for characterizing pharmacological or potentiallypharmacological target proteins.

This object is achieved by CNG channels composed of α3 subunits whichhave been modified in the position corresponding to threonine T537 inthe bovine α3 subunit so as to have higher sensitivity to cAMP and/orhigher selectivity for cAMP compared to cGMP in comparison with the wildtype according to SEQ ID NO 1 and 2.

These ion channels have a sensitivity to cAMP and cGMP similar to thatof the C460W/E583M mutant.

The invention moreover relates to a method for preparing said CNGchannels. The invention also relates to expression vectors comprisingthe nucleic acids for the modified CNG channels. The invention likewiserelates to cell lines which are transformed with the describedexpression vectors and which can express the CNG channels. Particularpreference is given to cell lines capable of coexpressing heterologouslyeither GPCRs, adenylate cyclases, phosphodiesterases or other proteinswhich regulate intracellular cAMP concentration together with a modifiedCNG channel.

The invention further relates to a method for preparing said cell lines,which comprises carrying out a transformation by means of expressionvectors. According to the invention, the genes for the proteins arepreferably cloned into the expression vector, followed by transformationof the cell lines.

According to the invention, preference is given to using CNG channelscomposed of α3 subunits. However, other subunits from bovine or otherorganisms are also suitable.

In the subunits used according to the invention, preference is given toreplacing the amino acid corresponding to threonine at position T537 inthe bovine α3 subunit with a different amino acid other than serine.Particular preference is given here to those subunits in which threoninehas been replaced with methionine or valine. SEQ ID NO 3 and 4 and,respectively, SEQ ID NO 5 and 6 depict the bovine α3 subunit as anexample of subunits modified in this way.

The ion channels of the invention are especially suitable as cellularcAMP sensors for measuring intracellular cAMP concentration. They arealso suitable for determining the action of ligands, agonists andantagonists on G protein-coupled receptors (GPCRs) which regulateintracellular cAMP concentration. Moreover, they may be used fordetermining the action of activators and inhibitors on adenylatecyclases and phosphodiesterases (effector proteins) which regulateintracellular cAMP concentration.

The invention further relates to the use of cellular measuring systemscomprising nucleic acids and the corresponding proteins for determiningthe action of chemical substances which influence the activity ofcellular components which regulate intracellular cAMP concentrationdirectly or indirectly.

The cellular measuring systems may be used universally and flexibly ascAMP sensors for pharmaceutical drug screening and drug characterizationand for characterization of pharmacologically relevant proteins. Thelatter include all G protein-coupled receptors, adenylate cyclases,phosphodiesterases and any other proteins involved in cAMP signalpathways.

Cellular cAMP sensors which may be prepared and used instead of theT537M mutant or the C460W/E583M mutant however, are in principle alsoother genetically modified CNG channels which have

-   (i) a similarly high or higher sensitivity to cAMP,-   (ii) a similarly high or higher sensitivity to cAMP or else-   (iii) a similarly high or higher sensitivity to and, in addition, a    similarly high    -   or higher selectivity for cAMP.

CNG channels of this kind may have (1) α3 subunits of other organisms,(2) other CNG-channel subunits from bovine or other organisms, and (3) ahomooligomeric or heterooligomeric composition of these subunits. Thesesubunits may (4) be genetically modified in each case at the positioncorresponding to position T537 in the α3 subunit of the bovine CNGchannel. The threonine in this position may have been replaced with amethionine or a valine or else with another amino acid (with theexception of serine). Such subunits may have (5) further geneticmodifications at other positions. These CNG channels may further (6)comprise chimeric subunits which have been genetically modified in thesame way at said position.

“Genetically modified at the position corresponding to position T537 inthe α3 subunit of the bovine CNG channel” means the following: thedifferent subunits of CNG channels (e.g. α1, α2, α3 and α4) or identicalsubunits of CNG channels of different organisms, such as, for example,the bovine and rat α3 subunits, have sequences which are highly similarto one another. Nevertheless, the position of the structurally andfunctionally important sections in the amino acid sequence of saidsubunits usually differs slightly. The skilled worker, however, is ableto identify the sequence sections and amino acids corresponding to oneanother by comparing the sequences. Threonine T537 in the binding sitefor cyclic nucleotides in the bovine α3 subunit, for example,corresponds to threonine T539 in the rat α3 subunit or to threonine T560in the bovine α1 subunit.

“Further genetic modifications” means that, in addition to amodifications of the invention, an amino acid has been replaced with adifferent one in at least one other position or an amino acid has beendeleted from or added to at least one other position.

“Chimeric subunits” means those CNG-channel subunits which are composedof at least two different subunit moieties, i.e., for example, a subunitcomposed of the amino-terminal moiety of the α1 subunit and thecarboxy-terminal moiety of the α3 subunit. Such chimeras can be readilyprepared by a skilled worker using molecular-biological methods and areoften utilized in order to combine particular properties of one proteinwith the properties of another protein or to transfer particularproperties to another protein or else to alter particular properties incomparison with the wild-type proteins. Chimeras of differentCNG-channel subunits have already been described (Seifert R., EismannE., Ludwig J., Baumann A., and Kaupp, U. B. (1999) Moleculardeterminants of a Ca²⁺-binding site in the pore of cyclicnucleotide-gated channels: S5/S6 segments control affinity of intraporeglutamates. EMBO J., 18, 119-130).

Six genes for subunits of CNG channels are known in vertebrates (α1-α4,β1, β2). Additionally, there exist different isoforms of these subunits(Sautter A., Zonh X., Hofmann F., and Biel M. (1998) An isoform of therod photoreceptor cyclic nucleotide-gated channel beta subunit expressedin olfactory. neurons. Proc. Natl. Acad. Sci. USA, 95, 4696-4701; BönigkW., Bradley J., Müller F., Sesti F., Boekhoff I., Ronnett G. V., KauppU. B., and Frings S. (1999) The native rat olfactory cyclicnucleotide-gated channel is composed of three distinct subunits. J.Neurosci., 19, 5332-5347). The α1 subunit (Kaupp U. B., Niidome T.,Tanabe T., Terada S., Bönigk W., Stühmer W., Cook N. J., Kangawa K.,Matsuo H., Hirose T., Miyata T., and Numa S. (1989) Primary structureand functional expression from complementary DNA of the rodphotoreceptor cyclic GMP-gated channel. Nature, 342, 762-766) and the β1subunit (Chen T. Y., Peng Y. W., Dhallam R. S., Ahamed B., Reed R. R.and Yau K. W. (1993) A new subunit of the cyclic nucleotide-gated cationchannel in retinal rods. Nature, 362, 764-767; Körschen H. G., IllingM., Seifert R., Sesti F., Williams A., Gotzes S., Colville C., MüllerF., Dose A., Godd M., Molday L., Kaupp U. Be., and Molday R. S. (1995) A240 kDa protein represents the complete beta subunit of the cyclicnucleotide-gated cation channel from rod photoreceptor. Neuron, 15,627-636) were first discovered in retinal rods, the α2 subunit (BönigkW., Altenhofen W., Müller F., Dose A., Illing M., Molday R. S., andKaupp U. B. (1993) Rod and cone photoreceptor cells express distinctgenes for cGMP-gated channels. Neuron, 10, 865-877) and the β2 subunit(Gerstner A., Zong X., Hofmann F., and Biel M. (2000) Molecular cloningand functional characterization of a new modulatory cyclicnucleotide-gated channel subunit from mouse retina. J. Neurosci., 20,1324-1332) in retinal cones, and the α3 subunit (Dhallan R. S., Yau K.W., Schrader K. A., and Reed R. R. (1990) Primary structure andfunctional expression of a cyclic nucleotide-activated channel fromolfactory neurons. Nature, 347, 184-187; Ludwig J., Margalit T., EismannE., Lancet D., and Kaupp U. B. (1990) Primary structure of cAMP-gatedchannel from bovine olfactory epithelium. FEBS Lett., 270, 24-29) andthe α4 subunit (Bradley J., Li J., Davidson N., Lester H. A., and ZinnK. (1994) Heteromeric olfactory cyclic nucleotide-gated channels: asubunit that confers increased sensitivity to cAMP. Proc. Natl. Acad.Sci. USA, 91, 8890-8894; Liman E. R. and Buck L. B. (1994) A secondsubunit of the olfactory cyclic nucleotide-gated channel confers highsensitivity to cAMP. Neuron, 13, 611-621) in olfactory cells of thenose.

In addition, CNG channels composed of said subunits were found innumerous other neuronal and non-neuronal cells and tissues (Richards M.J. and Gordon S. E. (2000) Cooperativity and cooperation in cyclicnucleotide-gated ion channels. Biochemistry, 39, 14003-14011). Moreover,CNG channels were found not only in vertebrates but also innonvertebrates such as, for example, in Drosophila melanogaster (BaumannA., Frings S., Godde M., Seifert R., and Kaupp U. B. (1994) Primarystructure and functional expression of a Drosophila cyclicnucleotide-gated channel present in eyes and antennae. EMBO J., 13,5040-5050) and plants (Leng Q., Mercier R. W., Yao W., and Berkowitz G.A. (1999) Cloning and first functional characterization of a plantcyclic nucleotide-gated cation channel. Plant Physiol., 121, 753-761).In principle, these and all other subunits of CNG channels can bemodified according to the invention.

The genetically modified CNG channels may be used in cellular testsystems as cAMP sensor for pharmacological studies,

-   (i) in order to study the action of ligands, agonists and    antagonists on membrane-bound G protein-coupled receptors (GPCRs)    which regulate intracellular cAMP concentration,-   (ii) in order to study the action of activators and inhibitors on    effector proteins (enzymes) which synthesize or hydrolyze cAMP,-   (iii) in order to study the action of activators and inhibitors on    other proteins which likewise intervene in the cAMP signal    transduction cascade in a regulating manner, but also-   (iv) in order to study the properties of GPCRs, effector proteins or    other proteins involved in cAMP signal transduction cascades.

The proteins referred to as “membrane-bound G protein-coupled receptors”(GPCRs) according to the invention belong to the phylogenetically mostvaried, extremely extensive family of membrane-bound receptors (overviewarticle: Morris A. J. and Malbon C. C. (1999) Physiological regulationof G protein-linked signaling. Physio. Rev., 79, 1373-1430): The familyof GPCRs probably comprises distinctly more than 1 000 different memberswhich can be classified on the basis of their sequence similarity(Probst W. C., Snyder L. A., Schuster D. I., Brosius J., and Sealfon S.C. (1992) Sequence alignment of the G-protein coupled receptorsuperfamily. DNA Cell Biol., 11, 1-20) or based on the chemical natureof their natural ligands. A compilation and classification of the GPCRsknown up to now can be found, for example, in the “GPCRDB” database(Horn F., Weare J., Beukers M. W., Horsch S., Bairoch A., Chen W.,Edvardsen O., Campagne F., and Vriend G. (1998) GPCRDB: an informationsystem for G protein-coupled receptors. Nucleic Acids Res., 26,275-279). Some of the representatives of the individual classes arelisted below in the form of an overview. Class A (“rhodopsin-like”)includes rhodopsin itself and different sequence-related receptors whichare classified on the basis of their natural ligands: these includereceptors for (1) biogenic amines such as, for example, the muscarinicacetylcholine receptors, adrenergic receptors, dopamine receptors,histamine receptors, serotonin receptors, octapamine receptors, for (2)peptides, such as, for example, the angiotensin receptors, chemokinereceptors, endotheline receptors, neuropeptide receptors, for (3)hormone proteins, such as, for example, FSH receptors, for (4) odorants,for (5) prostanoids, such as, for example, prostaglandin receptors, orfor (6) nucleotides, such as, for example, adenosine receptors. Class B(“secretin-like”) includes the secretin receptors themselves and, forexample, receptors for calcitonin, glucagon, diuretic hormones or CRF(corticotropin-releasing factor). Class C (“metabotropicglutamate/pheromones”) includes the metabotropic receptors themselvesand also GABA-B receptors and others. Further classes comprise receptorsfrom plants, fungi, insects, bacteria. All classes contain receptorswhose function is not yet known or whose natural ligand is not yet known(orphan receptors). The natural ligands of each of about 200 differentGPCR types are currently known and about a further 100 GPCR types areorphan GPCRs. 700 or more GPCRs are presumably activated by odorants ortastants. It is possible in principle for all GPCRs regulatingintracellular cAMP concentration to be coexpressed with the inventivegenetically modified CNG channels as cAMP sensor in heterologousexpression systems and for the action of ligands, agonists andantagonists to be studied pharmacologically.

It is also possible to study GCRPs which normally do not regulateintracellular cAMP concentration via stimulatory or inhibitory Gproteins. GPCRs of this kind may be altered by genetic modification insuch a way that they couple to the cAMP signal pathway. Said geneticmodification may be carried out, for example, by preparing chimericGCRPs (Liu J., Conklin B. R., Blin N., Yun J. and Wess J. (1995)Identification of a receptor-G-protein contact site critical forsignaling specificity and G protein aviation. Proc. Natl. Acad. Sci.USA, 92, 11642-11646).

“Agonists” and “ligands” refer to substances which activate GCRP.

In contrast, “antagonists” refer to substances which, although beingable to bind to GCRPs, cannot activate them. Antagonists inhibit theaction of ligands or agonists in a dose-dependent manner.

“Effector proteins” which regulate intracellular cAMP concentrationdirectly include adenylate cyclases and phosphodiesterases.

“Adenylate cyclases” whose activity is controlled in a GPCR-mediatedmanner are large, membrane-bound enzymes which catalyze the formation ofcAMP from Mg²⁺ adenosine triphosphate and which are present in mostcells, tissues and organs of the human body (Tang W. J. and Hurley J. H.(1998) Catalytic mechanism and regulation of mammalian adenylylcyclases. Mol. Pharmacol., 54, 231-240). Nine different classes of saidadenylate cyclases are known altogether. Adenylate cyclases of this kindare also endogenously expressed in the cellular test systems of theinvention and can be activated by heterologously expressed GPCRs andtherefore play a decisive part in the functioning of the test system.Another class comprises soluble adenylate cyclases whose activity ispresumably not regulated in a GPCR-mediated manner (Buck J., Sinclair M.L., Schapal L., Cann M. J., and Levin L. R. (2000) Cytosolic adenylylcyclase defines a unique signaling molecule in mammals. Proc. Natl.Acad. Sci. USA, 96, 79-84). It is possible in principle to coexpress alladenylate cyclases with the inventive genetically modified CNG channelsas cAMP sensor in heterologous expression systems and to studypharmacologically the action of inhibitors and activators.

“Phosphodiesterases” (PDEs) are enzymes inside the cell which hydrolyzecAMP and cGMP to give adenosine monophosphate (AMP) and guanosinemonophosphate (GMP), respectively (Francis S. H., Turko I. V., andCorbin J. D. (2000) Cyclic nucleotide phosphodiesterases: relatingstructure and function. Prog. Nucleic Acid Res. Mol. Biol., 65, 1-52).Eleven different types of PDEs are known altogether. Some of said PDEsspecifically hydrolyze cGMP, others in turn hydrolyze cAMP, and othersagain hydrolyze both cAMP and cGMP. Like GPCRs and adenylate cyclases,the PDEs are expressed in most cells, tissues and organs of the humanbody. In contrast to adenylate cyclases, however, only PDE6 which isspecific for photoreceptors is activated in a GPCR-mediated manner. Theactivity of the other PDEs is instead regulated by different othermechanisms. It is possible in principle to coexpress all PDEs whichhydrolyze cAMP with the inventive genetically modified CNG channels ascAMP sensor in heterologous expression systems and to studypharmacologically the action of inhibitors and activators.

“Activators” refers to substances which interact directly with adenylatecyclases or PDEs and thereby increase the enzymatic activity thereof.

“Inhibitors”, in contrast, refer to substances which likewise interactdirectly with adenylate cyclases or PDEs but which reduce the activitythereof.

It is possible in principle for all proteins which regulateintracellular cAMP concentration to be coexpressed with the inventivegenetically modified CNG channels as cAMP sensor in heterologousexpression systems and to be studied pharmacologically.

The proteins to be studied may be expressed in heterologous systemseither transiently or, preferably, stably.

“Transient expression” means that the heterologously expressed proteinis expressed by the cells of the expression system only for a definedperiod of time.

“Stable expression” means that the introduced gene is stably integratedinto the genome of the cells of the heterologous expression system. Thenew cell line produced in this way expresses the corresponding proteinin each subsequent cell generation.

For expression, the cDNA coding for the protein to be studied is clonedinto an expression vector and transformed into the cells of a suitableexpression system.

“Expression vectors” refer to any vectors which can be used forintroducing (“transforming”) cDNAs into the appropriate cell lines andfunctionally expressing there the corresponding proteins (“heterologousexpression”). Preferably, the transformation may be carried out usingthe pcDNA vectors (Invitrogen).

Suitable “heterologous expression systems” are in principle alleukaryotic cells such as, for example, yeast, Aspergillus, insect,vertebrate and in particular mammalian cells. Examples of suitable celllines are CHO (Chinese hamster ovary) cells, for example the K1 line(ATCC CCL 61), including the Pro 5 variant (ATCC CRL 1781), COS cells(African green monkey), for example the CV-1 ceu line (ATCC CCL 70),including the COS-1 variant (ATCC CRL 1650) and the COS-7 variant (ATCC1651), BHK (baby hamster kidney) cells, for example the line BHK-21(ATCC CCL 10), MRC-5 (ATCC CCL 171), murine L cells, murine NIH/3T3cells (ATCC CRL 1658), murine C127 cells (ATCC CRL-1616), humancarcinoma cells such as, for example, the HeLa line (ATCC CCL 2),neuroblastoma cells of the lines IMR-32 (ATCC CCL 127), neuro-2A cells(ATCC CLL 131), SK-N-MC cells (ATCC HTB 10) and SK-N-SH cells (ATCC HTB11), PC12 cells (ATCC CRL 1721), and Sf9 cells (Spodoptera frugiperda)(ATCC CRL 1711). Preference is given to using HEK 293 (human embryonickidney) cells (ATCC CRL 1573), including the SF variant (ATCC 1573.1).Particular preference is given to the cell line prepared according tothe invention, DSM ACC 2516.

The action of test substances on the protein to be studied can bemeasured using a fluorescence-optical measurement method.

The protein to be studied is heterologously expressed together with cAMPsensor of the invention in a cellular test system and activated orinhibited by ligands, agonists, antagonists, activators or inhibitors.The cAMP sensor registers the changes in cAMP concentration and Ca²⁺ions flow into the cell to a larger or reduced extent.

“Fluorescence-optical measurement methods” means that the change inintracellular Ca²⁺ concentration is made visible using a fluorescentCa²⁺ indicator. By now, a multiplicity of different Ca²⁺ indicators areknown (Haugland R. P., (1996) Handbook of fluorescent probes andresearch chemicals. Molecular Probes Inc.). They include, for example,Fluo-3, Calcium Green, Fura-2 and Fluo-4 (Molecular Probes), the latterbeing preferred according to the invention. Since indicators of thiskind are usually water-soluble and therefore cannot pass the hydrophobiclipid membrane of cells, the indicators are instead applied in the formof an acetoxy methyl ester compound (Tsien R. Y. (1981) A non-disruptivetechnique for loading calcium buffers and indicators into cell. Nature,290, 527-528). These compounds, in contrast, are hydrophobic and aretaken up by the cells. Inside the cell, the ester bond is cleaved byendogenous, intracellular esterases and the indicator is again presentin its water-soluble form in which it remains in the cell interior whereit accumulates and can thus be used as intracellular Ca²⁺ indicator.This indicator, when excited with light of a suitable wavelength, thenshows fluorescence which depend on the intracellular Ca²⁺ concentration.The degree (amplitude) of fluorescence and the time course (kinetics)correlate with the degree and the time course of activation of theprotein studied and can be monitored in real time using fluorescencedetectors with a very good signal-to-noise ratio and plotted using asuitable software.

Likewise, the aequorin protein complex which consists of apoaequorin andthe chromophoric cofactor coelenterazine or comparable complexes may beused as Ca²⁺ indicators for measuring intracellular Ca²⁺ concentration(Brini M., Pinton P., Pozzan T. and Rizzuto R. (1999) Targetedrecombinant aequorins: tools for monitoring (Ca²⁺) in the differentcompartments of a living cell. Micrsc. Res. Tech., 46, 380-389). Forthis purpose, apoaequorin must be heterologously expressed together withthe cAMP sensor of the invention and the protein to be studied in thecellular test system. Prior to the measurements, the cells must beincubated with coelenterazine so that apoaequorin and coelenterazine canassemble to give the active aequorin complex. When the intracellularCa²⁺ concentration increases, coelenterazine is oxidized tocoelenteramide. In this process, CO₂ is formed and luminescence isemitted. Disadvantageously, said process is irreversible. Saidluminescence may be registered using a suitable optical detector(luminescence-optical measurement method). Although this opticalmeasurement method has a similar sensitivity to the fluorescence-opticalmeasurement method, it is however less suitable for measurements inwhich the course of the reaction is followed in real time.

Fluorescence- or luminescence-optical measurements using a test systemof the invention may be carried out in cuvette measuring devices, inCa²⁺ imaging microscopes or in fluorescence or luminescence readers.

According to the invention, preference is given to carrying out themeasurements in wells of plastic containers (multiwell plates) influorescence readers. The cells may be introduced in suspension or else,preferably, attached to the bottom of said wells. Multiwell plateshaving a different number of wells may be used, such as, for example,multiwell plates having 96, 384, 1536 or more wells. Said multiwellplates make it possible to carry out a multiplicity of identical ordifferent measurements in a single plate.

“Fluorescence reader” or “luminescence reader” are very sensitiveoptical measuring devices which can be used to measure fluorescence orluminescence in multiwell plates. It is possible to study in suchdevices the action of ligands, agonists, antagonists, activators orinhibitors very rapidly and with a high throughput.

According to the invention, it is possible to study the properties ofGPCRs, effector proteins or other proteins involved in cAMP signaltransduction cascades quantitatively using fluorescence- orluminescence-optical measurements.

However, it is also possible in principle, according to the invention,to carry out measurements with a high throughput of tests per day. Thesearch for new pharmacological active compounds is thus possible. It ispossible here to test up to 100 000 substances per day (high throughputscreening, HTS screening) or more than 100 000 substances per day(Ultra-HTS screening, UHTS screening).

Using a FLIPR384 fluorescence reader (Molecular Devices), for example,it is possible to carry out up to 384 independent measurementssimultaneously and to monitor fluorescence in real time.

The invention is described in more detail below on the basis of theexemplary embodiments and the attached figures:

EXAMPLES 1-4

Genetically modified, homooligomeric bovine α3 CNG channels wereprepared, whose sequence has, in place of threonine T537 of thewild-type channel, a methionine (T537M) or valine (T537V).

For this purpose, the nucleic acid coding for the α3 subunit of thebovine CNG channel (SEQ ID NO 1) was altered in position 537 bysite-specific mutagenesis using molecular-biological methods(“genetically modified”) so that a nucleic acid coding for the T537Mmutant (SEQ ID NO 3) and another one coding for the T537V mutant (SEQ IDNO 5) were generated (see Methods).

The SEQ ID NO 2, 4 and 6 depict the amino acid sequences of thewild-type channel, the T537M mutant and the T537V mutant, respectively.

The sensitivity and selectivity of the T537M mutant and of the T537Vmutant for cAMP and cGMP were determined by electrophysiological methods(see Methods) and compared with the properties of the wild-type channel.For this purpose, the corresponding nucleic acids were cloned into theexpression vector pcDNA3.1 (Invitrogen) (see Methods). The expressionconstructs were then used to transform the cells of the human embryonickidney cell line 293 (HEK293 cells) and the corresponding proteins werefunctionally expressed therein either transiently or stably (seeMethods). The results of the studies are depicted in FIGS. 1-4.

The FIGS. 1A, 2A, 3A and 4A depict in each case the current-voltage (IV)relationship of the heterologously expressed genetically modified CNGchannels in the presence of different concentrations of cAMP (1A, 3A)and cGMP (2A, 4A), respectively. They depict in each case acurrent-voltage relationship typical for CNG channels in the presence ofcAMP and cGMP, respectively, and confirm that the channels arefunctionally expressed in the HEK293 cells.

FIGS. 1-4 B depict the dependence of the average current on the cAMPconcentration and cGMP concentration, respectively, in each case in theform of a dose-response relationship. The results of these measurementswere used to calculate the sensitivity of the CNG channels to cAMP andcGMP. The concentration of cAMP and cGMP with V_(m)=+100 mV, at whichsaid channels conduct half of the possible maximum current (K_(1/2)value) is used as measure for the sensitivity of said CNG channels. Thelower K_(1/2), the higher the sensitivity of the channel to thecorresponding cyclic nucleotide.

FIGS. 1B (T537M mutant) and 3B (T537V mutant) depict in each case thedose-response relationship for cAMP; FIGS. 2B (T537M mutant) and 4B(T537V mutant), on the other hand, indicate in each case thedose-response relationship for cGMP.

In FIGS. 1C (T537M mutant) and 3C (T537V mutant), in each case thenormalized dose-response relationship of the mutants is compared withthat of the α3 wild-type channel for cAMP; in FIGS. 2C (T537M mutant)and 4C (T537V mutant), on the other hand, in each case the normalizeddose-response relationship is compared with that of the α3 wild-typechannel for cGMP.

The table below summarizes the sensitivity properties of the geneticallymodified CNG channels and the wild-type channel. The K_(1/2) values forcAMP and for cGMP are listed.

K_(1/2) for cAMP K_(1/2) for cGMP 1. Wild type 80 μM 1.6 μM 2. T537S 14μM 0.7 μM 3. T537M 2.7 μM 14.9 μM 4. T537V 34 μM 241 μM

Half-maximum activation of the T537M mutant, the T357V mutant and thewild type is produced by 2.7 μM cAMP (FIG. 1C), 34 μM cAMP (FIG. 3C) and80 μM cAMP (FIGS. 1C, 3C), respectively. The T537M mutant and the T547Vmutant are about 30 times and 3 times, respectively, more sensitive tocAMP in comparison with the wild type. In comparison with the T537Smutant, the T537M mutant is about 5 times more sensitive to cAMP, whilethe T537V mutant is about 2.5 times less sensitive to cAMP than theT537S mutant.

Half-maximum activation of the T537M mutant, the T537V mutant and thewild type is produced by 14.9 μM cGMP (FIG. 2C), 241 μM cGMP (FIG. 4C)and 1.6 μM cGMP (FIGS. 2C, 4C), respectively. Thus, the T537M mutant andthe T537V mutant are about 9 times and about 150 times, respectively,less sensitive to cGMP in comparison with the wild type. In comparisonwith the T537S mutant, the T537M mutant is 21 times less sensitive tocGMP and the T537V mutant is even about 350 times less sensitive tocGMP.

The selectivity of a CNG channel for cAMP or cGMP is obtained bycomparing the K_(1/2) values. Example: The T537M mutant has a K_(1/2) of2.7 μM for cAMP and a K_(1/2) of 14.9 μM for cGMP. This means thathalf-maximum activation of said mutant is produced by as low as 2.7 μMcAMP but only by 14.9 μM cGMP. According to this, the mutant is markedlymore sensitive to cAMP than to cGMP. The quotient (14.9 μM/2.7 μM)indicates the relative selectivity. Thus, the T537M mutant is about 6times more selective for cAMP. The T537V mutant is likewise about 6times more selective for cAMP. The T537S mutant, on the other hand, isabout 20 times and the wild-type channel even about 50 times moreselective for cGMP.

The results of the measurements show the following: CNG-channel mutantswhose (1) absolute cAMP sensitivity is very much higher than that of thewild-type channels were generated and identified, and (2) theselectivity for cAMP and cGMP is reversed. The genetic modificationsimpart an enormous selective sensitivity to cAMP to these CNG channels.In addition, the two genetically modified CNG channels are soinsensitive to cGMP that even relatively large changes in intracellularcGMP concentration do not interfere with the cAMP concentrationmeasurement. Specifically, the T537M mutant which is preferred accordingto the invention is particularly suitable as cellular cAMP sensor. Itmay be used, for example, in cellular test systems for studying theaction of substances which can influence the intracellular cAMPconcentration and renders such test systems usable in practice in thefirst place: a sensor of this kind may then be used to register reliablyand with a very good signal-to-noise ratio and monitor in real time eventhose slight changes in intracellular cAMP concentration as aretriggered, for example, by the activation of GPCRs.

Methods

Preparation of Genetically Motivated CNG Channels By Site-SpecificMutagenesis

The cDNA for the α3 subunit of the bovine CNG channel was excised withthe aid of suitable restriction endonucleases (Eco RV and Nsi I) fromthe plasmid pCHOLF102 (Altenhof W., Ludwig J., Eismann E., Kraus W.,Bönigk W. and Kaupp U. B. (1991) Control of ligand specificity in cyclicnucleotide-gated channels from rod photoreceptors and olfactoryepithelium. Proc. Natl. Acad. Sci. USA, 88, 9868-9872) and cloned intopcDNAlamp (Invitrogen). The plasmid was referred to as pcA-bolf. ThecDNA fragment was then cloned via Eco RV and Xba I into pcDNA3derivatives. The plasmid was referred to as pc3-bolf. The geneticallymodified α3 subunit of the CNG channel was prepared by means ofsite-specific mutagenesis (Herlitze S. and Koenen M. (1990). A generaland rapid mutagenesis method using polymerase chain reaction. Gene, 91,143-147). The mutagenesis primer for preparing the T537M-α3 subunit hadthe following sequence (SEQ ID NO 7): 5′-CGACGCATGGCGAACATCCGCAGTCT-3′

The mutagenesis primer for preparing the T537V-α3 subunit had thefollowing sequence (SEQ ID NO 8): 5′-CGACGCGTCGCGAACATCCGCAGTCT-3′

First, a PCR of pc3-bolf was carried out using the mutagenesis primerand the counterprimer #1817 (5′-TTGGCTGCAGCTATTATGGCTTCTCGGCAG-3′) (SEQID NO 9). A 100-μl PCR mixture comprised 10 ng of template DNA, 1×PCRbuffer of Taq polymerase incl. 1.5 mM of MgCl₂, 200 μM of dNTPs, 1 U ofTaq polymerase and in each case 150 ng of the two primers. The PCRconditions were as follows. 2 minutes of denaturation at 94° C.,followed by 25 cycles of in each case 45 s at 94° C., 45 s at 46° C., 45s at 72° C. The 404 bp fragment was purified and used together with theoverlapping 666 bp Sma I/Pvu II restriction fragment as template for afurther PCR. The flanking primers #1813(5′-GTCGGATCCTCCACACTCAAGAAAGTG-3′) (SEQ ID NO 10) and #1817 were usedfor amplification. The PCR mixture had the same composition as indicatedabove, with the template used being 250 ng of the 1st PCR fragment and10 ng of the restriction fragment instead of 10 ng of plasmid DNA. ThePCR fragment was cleaved with BsrGl and substituted for thecorresponding fragment in pc3-bolf. The sequence of the altered DNAsection was checked by sequencing.

Isolation of cDNA Clones

The CRF receptor was cloned by carrying out a PCR of primary strand cDNAof rat hypophyses, using the primers #843(5′-AGCGGGATCCACCATGGGACGGCGCCCGCA-3′) (SEQ ID NO 11) and #842(5′-GGCCTGGAGCTCACACTG-3′) (SEQ ID NO 12). A 100 μl PCR mixturecomprised 10 ng of primary strand cDNA, 1×PCR buffer for Taq polymeraseincl. 1.5 mM of MgCl₂, 200 μM of dNTPs, 1 U of Taq polymerase and ineach case 150 ng of the two primers. The PCR conditions were as follows.2 minutes of denaturation at 94° C., followed by 44 cycles of in eachcase 45 s at 94° C., 45 s at 56° C., 75 s at 72° C. The 1271 bp fragmentwas cloned via BamHI and SacI in pBluescript SK⁻ into (pBCRFR1). Thesequence corresponds to the published sequence L25438 (Chang, C. P.,Pearse R. V. II, O'Connel S. and Rosenfed M. G. (1993). Identificationof a seven transmembrane helix receptor for corticotropin-releasingfactor and sauvagine in mammalian brain. Neuron, 11, 1187-1195). ThecDNA was subcloned into a pcDNA derivative for heterologous expression(pcCRFR1).

The plasmid pcK₁M which contains the cDNA of the dopamine receptor fromDrosophila (Gotzes F., Balfanz S. and Baumann A. (1994) Primarystructure and functional characterization of a Drosophila dopaminereceptor with high homology to human D1/5 receptors. Receptors Channels,2, 131-141), was kindly provided by Dr. F. Gotzes.

Heterologous Expression in HEK 293 Cells and Preparation of Stable CellLines

Transient expression in HEK 293 cells was carried out as described inBaumann A., Frings S., Godde M., Seifert R. and Kaupp U. B. (1994)Primary structure and functional expression of a Drosophila cyclicnucleotide-gated channel present in eyes and antennae. EMBO J., 13,5040-5050. For electrophysiological characterization, the cells weretransferred to glass slides which had been coated with poly L-lysine, onthe day after transfection. The electrophysiological studies were thencarried out on the following day.

To prepare the stable cell lines, the cells were transfected in the samemanner. Cell clones stably expressing the desired gene were selected byseeding 2×10⁴ cells on a 9 cm cell culture dish on the day aftertransfection. The cells were cultured for 20 days, with either G418 (800μg/ml) (Calbiochem), Zeocin (100 μg/ml) (Invitrogen) or Hygromycin (100μg/ml) (Invitrogen) being added to the cell culture medium. After 20days, the cell clones expressing the resistance gene were isolated andexpression of the CNG-channel gene or of the receptor gene was checkedby Western blot analyses and functional studies (electrophysiologicalmeasurement and fluorescence-optical measurements). For Western blotanalysis, the cells were homogenized in a lysis buffer (10 mM Hepes, 1mM DTT and 1 mM ETDA at pH 7.4), 5×shock-frozen (in liquid nitrogen) andfinally centrifuged at 55 000 rpm for 10 min. The membrane pellet wasresuspended in dissolving buffer (150 mM NaCl, 1 mM MgCl₂, 20 mM Hepesat pH 7.5, 0.1 mM EGTA and 0.5% Triton X-100). In each case 3 μg ofmembrane proteins were separated by means of SDS-PAGE, transferred toImmobilon membranes and labeled with specific antibodies. Theimmunoreactivity was made visible with the aid of the ECL detection kit(Amersham).

Double-stable cell lines were prepared by continuing culturing a cellclone which stably expressed either the CNG-channel gene or the receptorgene and using it for transfecting the in each case other cDNA. The cellclones were selected and chosen as described above.

The fluorescence-optical measurements partly involved using cells whichstably expressed a genetically modified α3 subunit of the CNG channeland transiently expressed the Drosophila dopamine receptor. For thispurpose, cells of the stable T537M cell line were transfected asdescribed by Baumann A., Frings S., Godde M., Seifert R., and Kaupp U.B. (1994) Primary structure and functional expression of a Drosophilacyclic nucleotide-gated channel present in eyes and antennae. EMBO J.,13, 5040-5050. On the day after transfection, the cells were seeded intoa multiwell plate with 96 wells. The cell density per well was 2×10⁴cells.

For the fluorescence-optical measurement of stable cell lines, the cellswere seeded into said multiwell plate with 96 wells one or two daysbefore the measurement. The cell density was from 1.5 to 4×10⁴ cells.

Electrophysiology

The genetically modified and the wild-type GNG channels were in eachcase heterologously expressed in HEK 293 cells (Baumann A., Frings S.,Godde M., Seifert R., and Kaupp U. B. (1994) Primary structure andfunctional expression of a Drosophila cyclic nucleotide-gated channelpresent in eyes and antennae. EMBO J., 13, 5040-5050). Theelectrophysiological characterization of the genetically modified CNGchannels was carried out using the patch clamp technique under voltageclamping conditions. The activation properties of the channels weredetermined and compared to those of the wild-type channels (FIGS. 1 to4):

inside-out patches were excised from cells which stably expressedgenetically modified or wild-type CNG channels. The bath solutioncontaining different concentrations of cyclic nucleotides was used toflow over the membrane patches. Starting from a holding voltage of 0 mV,sudden voltage changes were applied to different test voltages between−100 mV and +100 mV at the patch with a step width of 20 mV. Thecurrents were registered and analyzed using standard methods.

FIG. 1A depicts the current-voltage (IV) relationship of the depictsT537M-α3 subunit of the bovine CNG channel. The average currents (I) atdifferent cAMP concentrations are plotted as a function of the voltage(Vm): 0 μM (filled circles), 0.3 μM (open circles), 1 μM (filledtriangles), 3 μM (open triangles), 10 μM (filled squares), 30 μM (opensquares), 100 μM (filled diamonds).

FIG. 1B shows the dependence of the average currents on cAMPconcentration for the T537M-α3 subunit of the bovine CNG channel at avoltage of +100 mV (filled circles). The continuous line has beencalculated according to the Hill equationI=(I_(max)−I_(min))c^(n)/(K^(n)+c^(n))+I_(min) (I: current; I_(max):maximum current; I_(min): minimum current; K_(1/2): concentration atwhich half-maximum activation of the channels occurs; n: Hillcoefficient; c: cAMP concentration) with the following parameters:I_(max)=328 pA; I_(min)=24 pA; K_(1/2)=2.9 μM; n=2.4.

Figure C depicts the normalized dose-response relationship of theT537M-α3 subunit of the bovine CNG channel (continuous line) and that ofthe wild-type bovine α3 subunit (dotted line) at +100 mV. The continuousand dotted lines have been calculated according to the normalized Hillequation I_(norm.)=c^(n)/(c^(n)+K^(n)) with the following averagedparameters: (continuous line: K_(1/2)=2.7 μM; n=2.4; dotted line:K_(1/2)=80 μM; n=2.0).

FIG. 2A depicts the current-voltage (IV) relationship of theheterologously expressed T537M-α3 subunit of the bovine CNG channel. Theaverage currents (I) at different cGMP concentrations are plotted as afunction of the voltage (Vm): 0 μM (filled circles), 1 μM (opencircles), 3 μM (filled triangles), 10 μM (open triangles), 30 μM (filledsquares), 100 μM (open squares), 300 μM (filled diamonds), 2 000 μM(open diamonds).

FIG. 2B shows the dependence of the average currents on cGMPconcentration for the T537M-α3 subunit of the bovine CNG channel at avoltage of +100 mV (filled circles). The continuous line has beencalculated according to the Hill equationI=(I_(max)−I_(min))c^(n)/(K^(n)+c^(n))+I_(min) (I: current; I_(max):maximum current; I_(min): minimum current; K_(1/2): concentration atwhich half-maximum activation of the channels occurs; n: Hillcoefficient; c: cGMP concentration) with the following parameters:I_(max)=167 pA; I_(min)=11 pA; K_(1/2)=12.0 μM; n=2.0.

FIG. 2C depicts the normalized dose-response relationship of theT537M-α3 subunit of the bovine CNG channel (continuous line) and that ofthe wild-type bovine α3 subunit (dotted line) at +100 mV. The continuousand dotted lines have been calculated according to the normalized Hillequation I_(norm.)=c^(n)/(c^(n)+K^(n)) with the following averagedparameters: (continuous line: K_(1/2)=14.9 μM; n=1.9; dotted line:K_(1/2)=1.6 μM; n=2.0).

FIG. 3A depicts the current-voltage (IV) relationship of theheterologously expressed T537V-α3 subunit of the bovine CNG channel. Theaverage currents (I) at different cAMP concentrations are plotted as afunction of the voltage (Vm): 0 μM (filled circles), 3 μM (opencircles), 10 μM (filled triangles), 30 μM (open triangles), 100 μM(filled squares), 300 μM (open squares), 1 000 μM (filled diamonds).

FIG. 3B shows the dependence of the average currents on cAMPconcentration for the T537V-α3 subunit of the bovine CNG channel at avoltage of +100 mV (filled circles). The continuous line has beencalculated according to the Hill equationI=(I_(max)−I_(min))c^(n)/(K^(n)+c^(n))+I_(min) (I: current; I_(max):maximum current; I_(min): minimum current; K_(1/2): concentration atwhich half-maximum activation of the channels occurs; n: Hillcoefficient; c: cAMP concentration) with the following parameters:I_(max)=181 pA; I_(min)=11 pA; K_(1/2)=23.4 μM; n=2.2.

FIG. 3C depicts the normalized dose-response relationship of theT537V-α3 subunit of the bovine CNG channel (continuous line) and that ofthe wild-type bovine α3 subunit (dotted line) at +100 mV. The continuousand dotted lines have been calculated according to the normalized Hillequation I_(norm.)=c^(n)/(c^(n)+K^(n)) with the following averagedparameters: (continuous line: K=34 μM; n=1.5; dotted line: K_(1/2)=80μM; n=2.0).

FIG. 4A depicts the current-voltage (IV) relationship of theheterologously expressed T537V-α3 subunit of the bovine CNG channel. Theaverage currents (I) at different cGMP concentrations are plotted as afunction of the voltage (Vm): 0 μM (filled circles), 30 μM (opencircles), 100 μM (filled triangles), 300 μM (open triangles), 1 000 μM(filled squares), 2 000 μM (open squares).

FIG. 4B shows the dependence of the average currents on cGMPconcentration for the T537V-α3 subunit of the bovine CNG channel at avoltage of +100 mV (filled circles). The continuous line has beencalculated according to the Hill equationI=(I_(max)−I_(min))c^(n)/(K^(n)+c^(n))+I_(min)(I: current; I_(max):maximum current; I_(min): minimum current; K_(1/2): concentration atwhich half-maximum activation of the channels occurs; n: Hillcoefficient; c: cGMP concentration) with the following parameters:I_(max)=1 389 pA; I_(min)=13 pA; K_(1/2)=255.2 μM; n=2.2.

FIG. 4C depicts the normalized dose-response relationship of the bovineT537V-α3 subunit (continuous line) and that of the wild-type bovine α3subunit (dotted line) at +100 mV. The continuous and dotted lines havebeen calculated according to the normalized Hill equationI_(norm.)=c^(n)/(c^(n)+K^(n)) with the following averaged parameters:(continuous line: K=241 μM; n=2.1; dotted line: K_(1/2)=1.6 μM; n=2.0).

Fluorescence-Optical Measurements

The fluorimetric measurements of intracellular Ca²⁺ concentrations werecarried out in multiwell plates with 96 wells (FIG. 5A-5G). The cellswere loaded with the Ca²⁺-sensitive fluorescent dye Fluo-4 one hourbefore the measurement. The loading solution contained 120 mM of NaCl, 3mM of KCl, 50 mM of glucose, 10 mM of Hepes (pH 7.4), 3 mM of MgCl₂ and4 μM of Fluo4-AM (molecular probes). The measuring solution contained120 mM of NaCl, 3 mM of KCl, 50 mM of glucose, 10 mM of Hepes (pH 7.4)and 3 mM of CaCl₂. The time course of the change in fluorescenceintensity after application of agonists, antagonists, enzyme activatorsor enzyme inhibitors was registered using a fluorescence reader(FLUOstar, BMG-Labtechnologies). The excitation wavelength was 485 nm.The emission wavelength was 520 nm.

EXAMPLE 5

FIG. 5A depicts the fluorimetrically measured change in intracellularCa²⁺ concentration in cells transiently expressing the Drosophiladopamine receptor (DR) (Gotzes F., Balfanz S., and Baumann A. (1994)Primary structure and functional characterization of a Drosophiladopamine receptor with high homology to human D1/5 receptors. ReceptorChannels, 2, 131-141) and stably expressing the T537M mutant of theα3-CNG channel. The time course of fluorescence intensity (RFU: relativefluorescence unit) after stimulation of the cells with differentconcentrations of the agonist dopamine is shown. The arrow marks thepoint in time at which the cells were stimulated with dopamine. Thedopamine-induced activation of the dopamine receptor first leads in thecell to activation of a stimulatory G protein and finally to activationof an adenylate cyclase which synthesizes cAMP. Binding of cAMP opensCNG channels and Ca²⁺ ions flow into the cell. The speed and extent ofthe change in Ca²⁺ concentration depend on the dopamine concentration.The dopamine concentration was 25 nM (filled circles), 50 nM (opencircles), 400 nM (filled triangles) or 600 nM (open triangles). Forcomparison, cells which do not express the dopamine receptor were alsostimulated with 25 nM dopamine (filled rectangles).

EXAMPLE 6

FIG. 5B depicts the fluorimetrically measured change in intracellularCa²⁺ concentration in cells which stably express both thecorticotropin-releasing factor (CRF) receptor and the T537M mutant ofthe α3-CNG channel. The time course of fluorescence intensity afterstimulation of the cells with the agonist CRF is shown. The arrow marksthe time at which the cells were stimulated with CRF. CRF-inducedactivation of the CRF receptor leads in the cell first to activation ofa stimulatory G protein and finally to activation of the adenylatecyclase which synthesizes cAMP (see, for example: Eckart K., RadulovicJ., Radulovic M., Jahn O., Blank T., Stiedl O., and Spiess J. (1999)Actions of CRF and its analogs. Curr. Med. Chem., 6, 1035-1053; PerrinM. H. and Vale W. W. (1999). Corticotropin releasing factor receptorsand their ligand family. Ann. N.Y. Acad. Sci., 885, 312-328). Binding ofcAMP opens CNG channels and Ca²⁺ ions flow into the cell. The speed andextent of the change in Ca²⁺ depend on the CRF concentration. The CRFconcentration was 100 pM (filled triangles), 300 pM (open circles) or 1000 pM (filled circles).

FIGS. 5A and 5B depict by way of example for two different GCRPscoupling to a stimulatory G protein that the method of the invention canbe used to measure the action of agonists on said GCRPs with highsensitivity. The method is equally suitable for all other GCRPs couplingto stimulatory G proteins. The examples of FIGS. 5A and 5B also showthat it is possible to express for the method of the invention theheterologously expressed proteins (genetically modified α subunit of theCNG channel and GCRP) both transiently and stably in the cells.

EXAMPLE 7

FIG. 5C indicates that the method is suitable for quantitativelydetermining the effectiveness of different antagonists. Cells whichstably express the corticotropin-releasing factor (CRF) receptor and theT537M mutants of the α3-CNG channel were treated with the helicalantagonist 9-41 (Rivier J., Rivier C., and Vale W. W. (1984) Syntheticcompetitive antagonists of corticotropin-releasing factor: effect onACTH secretion in the rat. Science, 224, 889-891), before beingstimulated with CRF (1 nM). The time course of fluorescence intensity isshown. The arrow marks the point in time at which the cells werestimulated with CRF. With the same CRF concentration, the speed andextent of the change in Ca²⁺ depend on the concentration of theantagonist. The concentration of the antagonist was 0 nM (filledcircles), 10 nM (open circles) or 100 nM (filled triangles).

EXAMPLE 8

FIG. 5D indicates that the method is suitable for measuring theeffectiveness of particular enzyme activators. The effect of anactivator of adenylate cyclase (AC) on the change in intracellular Ca²⁺concentration is shown. Cells which stably express the T537M mutants ofthe α3-CNG channel were stimulated with different concentrations of theAC activator forskolin. The time course of fluorescence intensity isshown. The arrow marks the point in time at which the cells werestimulated with forskolin. Forskolin directly activates the adenylatecyclase endogenous to said cells (Seamon K. B. and Daly J. W. (1981)Forskolin: a unique diterpene activator of cyclic AMP-generatingsystems. J. Cyclic Nucleotide Res., 7, 201-224).

The rate and extent of the change in intracellular Ca²⁺ concentrationdepend on the concentration of the AC activator. The forskolinconcentration was 0.5 μM (open triangles), 0.75 μM (filled triangles), 2μM (open circles) or 4 μM (filled circles).

EXAMPLE 9

FIG. 5E indicates that the method is suitable for determining theeffectiveness of particular enzyme inhibitors. The effect of aphosphodiesterase (PDE) inhibitor on the forskolin-induced change inintracellular Ca²⁺ concentration is shown. Cells which stably expressthe T537M mutants of the α3-CNG channel were first treated withdifferent concentrations of the PDE inhibitor IBMX and then stimulatedwith 5 μM forskolin. The plot shows the time course of fluorescenceintensity. The arrow marks the point in time at which the cells werestimulated with forskolin. The PDE inhibitor inhibits the activity ofthe endogenous PDEs and thereby reduces degradation of cAMP synthesizeddue to a stimulation of the endogenous AC. The extent of the change inintracellular Ca²⁺ concentration depend on the concentration of the PDEinhibitor. The IBMX concentration was 0 μM (filled circles), 10 μM (opencircles), 50 μM (filled triangles) or 100 μM (open triangles).

The example shows that the method is suitable for measuring sensitivelythe effectiveness of PDE inhibitors. It is possible to study inhibitorsof endogenous PDEs but also inhibitors of heterologously expressed PDEs.

The method of the invention is also suitable for determining theactivity of GCRPs coupling to the inhibitory G protein G_(i). If GCRPsof this kind are activated, the cellular cAMP concentration is reducedbecause ACs are inhibited. For the method of the invention, the cellsmust first be stimulated to synthesize cAMP (e.g. by the AC activatorforskolin), before the activity of agonists can then be measured.Simultaneous or sequential application of antagonists and agonists makeit also possible to test the effectiveness of antagonists for such GCRP.

The method is analogously suitable for determining any substancescapable of increasing or reducing the cAMP concentration in the cell inany way. The direct points of attack of these substances (e.g. GCRP orenzymes) may either be present endogenously in the cells or may betransiently or stably expressed in cells together with geneticallymodified CNG channels.

EXAMPLE 10

FIG. 5F indicates that the method makes also quantitative analysespossible. Cells which stably express an endogenous adenosine receptor(Cooper J., Hill S. J. and Alexander S. P. (1997). An endogenous A2Badenosine receptor coupled to cyclic AMP generation in human embryonickidney (HEK 293) cells, Br. J. Pharmacol., 122, 546-550)and the T537Mmutant of the α3-CNG channel were stimulated with differentconcentrations of adenosine. The time course of fluorescence intensityis shown. The arrow marks the point in time at which the cells werestimulated with adenosine. The adenosine-induced activation of theadenosine receptor leads in the cell first to activation of astimulatory G protein and finally to activation of the adenylate cyclasewhich synthesizes cAMP. Binding of CAMP opens CNG channels and Ca²⁺ ionsflow into the cell. The extent and rate of the change in intracellularCa²⁺ concentration depend on the adenosine concentration. The adenosineconcentration was 0.9 μM (filled circles) and 1.5 μM (open circles), 3μM (filled triangles), 6 μM (open triangles), 25 μM (filled rectangles)or 37.5 μM (open rectangles).

FIG. 5G shows the dose-response relationship for adenosine. The rate ofthe change in fluorescence after addition of adenosine (initial rate)correlates with the adenosine concentration. The initial rates areplotted as a function of adenosine concentration. The continuous linehas been calculated according to the Hill equation: initial slope=a×c^(n)/(K^(n)+c^(n))_(n) (c=adenosine concentration; K: concentrationat which the half-maximum initial rate is reached; n: Hill coefficient;a =maximum initial slope) with the following parameters: a=1 403 RFU/s;K=5.15 μM; n=2.16), The K_(1/2) value determined in this way agrees verywell with the value of 5 μM indicated in the literature (Peakman N. C.and Hill S. J. (1994) Adenosine A2B receptor-mediated cyclic AMPaccumulation in primary rat atrocytes, Br. J. Pharmacol., 111, 191-198).

EXAMPLE 11

FIG. 5H demonstrates the enormous improvement in sensitivity of themeasuring system due to the use of the modified subunit of the α3-CNGchannel. Cells which stably express the T537M mutant of the α3-CNGchannel were stimulated with 3 μM adenosine to about half maximum (opencircles) or with 25 μM adenosine to maximum (filled circles). The extentof the change in intracellular Ca²⁺ concentration (fluorescence signal)was plotted as a function of time. The arrow marks the point in time atwhich the cells were stimulated with adenosine. Even after stimulationwith half-maximum adenosine concentration a distinct increase in thefluorescence signal can be observed. In contrast, the cells which stablyexpress the wild-type α3-CNG channel show no increase in thefluorescence signal, even after maximum stimulation with 25 μM adenosine(filled triangles). Cells which stably express the T537S mutant of the.alpha.3-CNG channel were also stimulated for comparison. After maximumstimulation of the endogenous adenosine receptor, only a very lowincrease in the fluorescence signal can be observed (filled rectangles).

The use of the genetically modified subunits of the inventionconsiderably increases the sensitivity of the intracellular test systemfor studying the action of substances influencing the cAMP signalpathway, only thereby making cell sensitivity usable in practice. Thesignals are so large and the signal-to-noise ratio is so good that evenquantitative analyses are possible. Suitable for preparing the celllines of the invention are (not only HEK 293 cells) but in principle anyeukaryotic cells which can be cultured in cell cultures. Both adherentlygrowing cells and cells growing in suspensions may be used.

1. A genetically modified cyclic nucleotide-gated ion channel (CNGchannel) which comprises a bovine α₃ subunit which has been modified ata binding site for cyclic nucleotides in the position corresponding tothreonine T537 in the bovine α₃ subunit, wherein threonine is replacedby methionine or valine, so that it has higher sensitivity for cAMP andhigher selectivity for cAMP than for cGMP in comparison with the wildtype bovine α3 subunit according to SEQ ID NO:2.
 2. A geneticallymodified vertebrate cyclic nucleotide-gated ion channel (CNG channel)which comprises a bovine α₃ subunit which has been modified at a bindingsite for cyclic nucleotides in the position corresponding to threonineT537 in the bovine α₃ subunit, wherein threonine is replaced bymethionine or valine, so that it has higher sensitivity for cAMP andhigher selectivity for cAMP than for cGMP in comparison with the wildtype bovine α3 subunit according to SEQ ID NO:
 2. 3. The geneticallymodified vertebrate cyclic nucleotide-gated ion channel (CNG channel)defined in claim 1 wherein threonine is replaced by methionine.
 4. Thegenetically modified vertebrate cyclic nucleotide-gated ion channel (CNGchannel) defined in claim 1 wherein threonine is replaced by valine. 5.A method for preparing a genetically modified CNG channel whichcomprises a bovine α₃ subunit which has been modified at a binding sitefor cyclic nucleotides in the position corresponding to position T537 inthe bovine α₃ subunit so that it has higher sensitivity for cAMP andhigher selectivity for cAMP than for cGMP in comparison to the wild typebovine α3 subunit according to SEQ ID NO:2, which comprises the step ofreplacing in the binding site of the bovine α₃ subunit, the amino acidcorresponding to threonine 537 in the bovine α₃ subunit with methionineor valine.
 6. A method for measuring intracellular cAMP concentration ina cell line, which comprises the steps of: (a) transforming the cellline with a vector capable of expressing the genetically modified cyclicnucleotide gated ion channel defined in claim 1; (b) measuring anelectric current across the cell line at the cell membrane; and (c)relating the electric current measured across the cell line at the cellmembrane to intracellular cAMP concentration.
 7. A method for measuringintracellular cAMP concentration in a cell line, which comprises thesteps of: (a) transforming the cell line with a vector capable ofexpressing the genetically modified cyclic nucleotide gated ion channeldefined in claim 1; (b) loading the cell line with a Ca²⁺ sensitivefluorescent dye; and (c) following step (b) measuring fluorescentintensity of the cell line; and (d) relating the measurement of thefluorescent intensity in the cell line to the concentration of Ca²⁺passing through the cell membrane into the cytoplasm and to theintracellular cAMP concentration.